Table 1. Expression systems used to produce membrane proteins, including benefits and drawbacks.

Expression system	Benefits	Drawbacks	Comments
Escherichia coli (manual induction)	Cheap, well-established technology, minimal equipment needed, effective for many bacterial targets.	Ineffective for most eukaryotic MPs.	Common strains include BL21(DE3), C41(DE3) & C43(DE3) [23], C44(DE3) & C45(DE3) [20] Lemo21(DE3) [19], BL21(DE3) containing either Rosetta2 or Origami.
Escherichia coli (auto-induction)	As manual induction except avoids need to monitor OD to add inducer.	Induction occurring after exponential phase can impair expression of some proteins.	Common strains used include many of those listed above. Autoinduction methods described by Studier [24].
Bacillus subtilis	Effective for secretion of (non-membrane) proteins into growth medium. Gram positive.	Less well-established than E. coli.	One of the original paper describing the use of B. subtilis [17].
Lactococcus lactis	Improved folding of eukaryotic membrane proteins over E. coli	Less well-established than E. coli.	Methods for protein production recently described [15, 18].
Saccharomyces cerevisiae	Improved expression of eukaryotic MPs.	Expression levels lower than Pichia.	Method that increases MP yield in S. cerevisiae [25].
Pichia pastoris	Improved folding and PTM of eukaryotic MPs; higher expression levels than S. cerevisiae.	Bottleneck due to need to screen many clones. Less suitable for HTP.	Use of P. pastoris for MP production [26].
Insect (Spodoptera frugiperda & Trichoplusia ni)	Improved folding and PTM of mammalian MPs over yeast, yield higher than mammalian cells.	Several weeks needed to generate baculovirus, more expensive than microbial systems. Cell culture lab and expertise needed.	Sf9, Sf21, Hi5, ExpiSf cell lines have been used to express MPs [27, 28].
Mammalian — transient	Ideal for correct folding and PTM of some eukaryotic MPs. Transfection is simple – no need for virus production or cloning/screening.	More expensive than microbial systems. Cell culture lab and expertise needed. Low yields. Large amounts of transfection-grade plasmid DNA needed for scale-ups.	Recent protocol production eukaryotic MP in Human embryonic kidney (HEK) cells [29]. An automated transient approach [5].
Mammalian — BacMam	Ideal for correct folding and PTM of some eukaryotic MPs. Useful for large-scale production.	More expensive than microbial systems. Cell culture lab and expertise needed. Low yields. Virus production time consuming.	Recent protocol describing use of the BacMam system for MP production [30].
Mammalian — stable	Ideal for correct folding and PTM of some eukaryotic MPs. Avoids requirement for large amounts of DNA or virus.	More expensive than microbial systems. Cell culture lab/ expertise needed. Low yields. Slower than transient. Lentiviral systems require containment at early stages.	Recent lentiviral protocol [31]. An automated stable approach [5].
Cell-free	Expression of highly toxic proteins possible. MP directly incorporated into encapsulation agents.	Cost prohibitive if large amounts needed.	Cell-free systems have been adapted from yeast, wheatgerm, insect and mammalian expression hosts [3, 32]. Also adapted for automation [6].